Inactive Smart

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Taking Orders from Light: Photo-Switchable Working/Inactive Smart Surfaces for Protein and Cell Adhesion Junji Zhang,* Wenjing Ma, Xiao-Peng He,* and He Tian Key Laboratory for Advanced Materials & Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China S Supporting Information *

ABSTRACT: Photoresponsive smart surfaces are promising candidates for a variety of applications in optoelectronics and sensing devices. The use of light as an order signal provides advantages of remote and noninvasive control with high temporal and spatial resolutions. Modification of the photoswitches with target biomacromolecules, such as peptides, DNA, and small molecules including folic acid derivatives and sugars, has recently become a popular strategy to empower the smart surfaces with an improved detection efficiency and specificity. Herein, we report the construction of photoswitchable self-assembled monolayers (SAMs) based on sugar (galactose/mannose)-decorated azobenzene derivatives and determine their photoswitchable, selective protein/cell adhesion performances via electrochemistry. Under alternate UV/vis irradiation, interconvertible high/low recognition and binding affinity toward selective lectins (proteins that recognize sugars) and cells that highly express sugar receptors are achieved. Furthermore, the cis-SAMs with a low binding affinity toward selective proteins and cells also exhibit minimal response toward unselective protein and cell samples, which offers the possibility in avoiding unwanted contamination and consumption of probes prior to functioning for practical applications. Besides, the electrochemical technique used facilitates the development of portable devices based on the smart surfaces for on-demand disease diagnosis. KEYWORDS: photoswitching, self-assembled monolayer, azobenzene, monosaccharide protein recognition, cell adhesion

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INTRODUCTION Light-responsive materials are attracting an increasing interest due to their noninvasive, intrinsically clean nature and high spatial/temporal resolution, which facilitates accurate and remote manipulations.1−4 Up until now, the majority of research on photocontrollable molecular operations has been achieved in the solution phase,5,6 in which molecules distribute and orient randomly. With an aim of grafting photoswitchable molecules to surfaces in order to facilitate the development of practical devices, we set out to develop photoresponsive “smart surfaces” that can “take orders” from remote light signals to carry out designated tasks.7−11 In the past decade, smart surfaces with photocontrollable systems for capture/release operations, electrocatalysis and wettability maneuvering via host−guest,12,13 and electrostatic14,15 as well as, simply, geometric effect of isomerization16−18 have been constructed and have played an important role as the bridge between photochromic molecules and optoelectronic devices. In © XXXX American Chemical Society

addition to optoelectronics, the development of smart surfaces grafted with biological ligands (e.g., nucleotides, peptides, and glycoligands) has also become an ever-blooming research area, because of their fascinating mimicking of essential life processes such as protein−ligand recognition,19−22 enzyme cascade catalysis,23,24 bacteria killing,25−27 and cell−cell adhesion.28−32 Combining bioactive ligands with photoresponsive molecules permits a new generation of surface materials with advanced functions and smart operations; e.g., the “ON−OFF” switchable molecular motives enable a recycling of the smart functional surfaces33,34 and reversible cell adhesion.35−38 In those previous studies, optical and mechanical spectroscopies including infrared reflection absorption spectroscopy,33 fluorescence spectroscopy,37,38 single-cell force spectroscopy,36 and Received: December 5, 2016 Accepted: February 21, 2017 Published: February 21, 2017 A

DOI: 10.1021/acsami.6b15599 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. (A) Structure of the Galactose-Azobenzene (Gal-Azo-SH) and Mannose-Azobenzene (Man-Azo-SH); (B) Photoswitching of the Smart Surface between Dormant (Inactive) and Activated (Working) States Towards Selective Protein/Cell Adhesion

other biological assays such as OD bacterial growth assay34 were applied as the means to monitor the photocontrollable procedure of the functionalized surfaces. Although efficient photoregulation of protein/cell adhesion and bacteria growth were achieved, bulky and expensive instruments and long detection time were required. Herein, we present sugarazobenzene functionalized smart surfaces to operate photocontrollable recognition and adhesion activities toward specific lectins (sugar-recognition proteins) and cancer cells that express sugar receptors (Scheme 1). In our system, electrochemistry techniques (cyclic voltammetry, differential pulse voltammetry, and Faradaic impedance spectrometry) were used to determine the photoswitchable performances of the smart surfaces. Compared with the above-mentioned methods, electrochemistry shows its advantage in terms of detection speed and accuracy, as well as the use of miniaturized devices (only a small potentiostat in connection with a personal laptop is needed), which has been extensively used for potable, ondemand environmental and biological analyses. Moreover, another advantage of using these smart surfaces is that the functional surfaces could be maintained in a dormant or shutdown state under certain light signal (OFF state) until being aroused by an activation light signal (ON state). Taking advantage of this feature and the noninvasive nature of light, a clean control of the unnecessary consumption or unwanted contamination during the material storage and delivery would be achieved based on our photocontrollable surface design. Glycosylation of fluorescence probes and smart surfaces has recently proven to be an efficient means to lower their cytotoxicity and increase the targeting ability toward specific lectins or cells/pathogens that express sugar receptors.39−51 In this study, two monosaccharides were used; galactose, which can selectively bind with peanut agglutinin (PNA) and interact with the asialoglycoprotein receptor (ASGPr) of Hep-G2 cells, and mannose, which can selectively bind with Conconavalin (Con A) and interact with the mannose receptor (MR) of macrophage cells.42−47 Galactosyl-/mannosyl-tethered azobenzene ligands were then synthesized and modified on gold

electrodes (Schemes S1 and S2, Figures S1 and S2) to operate a switchable ON−OFF surface activity under alternate UV/vis irradiation. We discuss the electrochemical features of the resulting monolayer-modified surfaces upon UV/vis light irradiation and the photoswitchable activation of the selective recognition and adhesion of lectins and cells.

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EXPERIMENTAL SECTION

General. All reagents and chemicals are commercially available. Proteins were purchased from Sigma-Aldrich. The detailed synthetic procedures and compound characterization are shown in Supporting Information. 1H and 13C NMR were recorded on NMR spectrometer (Bruker AvanceIII 400 and 100 MHz) in CDCl3 solutions with tetramethylsilane (TMS) as the internal standard. UV−vis spectra were recorded on a Varian Cray 60 spectrophotometer. Highresolution mass spectra were recorded using a Waters LCT Premier XE spectrometer. Time of flight secondary ion mass spectrometry (TOF-SIMS) experiments were performed using a TOF-SIMS V spectrometer (IONTOF GmbH, Münster, Germany). A pulsed 30 keV Bi3+ ion beam was used as the primary ion beam for all measurements. The analysis area was 100 × 100 μm. All data were obtained and analyzed using the IONTOF instrument software. Mass spectra were calibrated using C+, CH+, CH2+, and Au3+ peaks in the positive mode and C−, CH−, CH2−, C2−, and Au2− peaks in the negative mode. The UV irradiation of Azo-SAMs was carried out by using a 365 nm UV handheld lamp (UVP, Inc., 115 V, 0.16 A), and the visible light irradiation was carried out by using a xenon lamp (LEO, Shenzhen, China) filtered with an optical filter (450−700 nm, Orient KOJI instrument Co., Ltd., Tianjin, China). All electrochemical experiments were conducted with a computer-controlled CHI 1220b electrochemical station (Chenhua Co., Ltd., Shanghai, China). Working electrodes were 3 mm diameter disk gold (Au) electrodes, used in conjunction with a Pt auxiliary electrode, and a saturated calomel electrode (SCE) in saturated KCl solution worked as the reference electrode. All voltammetric experiments were performed after deaeration for 5 min with nitrogen. All experiments were carried out at room temperature. Monolayer Formation. The Au electrode was first polished on an emery paper and alumina slurry until a mirror-like surface was obtained. Sonication in ethanol solution for ∼3 min was followed. The B


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Figure 1. TOF-SIMS spectra of (A) Gal-Azo SAM and (B) Man-Azo SAM. polished gold electrodes were chemically oxidized by submerging them in 10 mL of hot “piranha” solution (98% H2SO4/30% H2O2 = 3:1 (v/ v)) in an open glass container for 10 min to remove the possible adsorbed contaminants. The gold electrodes were then rinsed extensively with copious amounts of Milli-Q water over 30 s. Electropolishing was then carried out with consecutive cyclic voltammograms in 0.5 mol/L sulfuric acid (potential range, −0.30 to 1.55 V vs SCE; 20 scans at a scan rate of 4 V s−1 and a sample interval of 0.001 V, followed by 4 scans at a scan rate of 0.1 V s−1 and a sample interval of 0.001 V) until a typical voltammogram for Au (the characteristic single, sharp gold oxide reduction peak is located at ∼0.9 V vs SCE, and multiple overlapping oxidation peaks appear in the range of 1.2−1.5 V vs SCE) was obtained. Because thiols are rather prone to air oxidation, the fabrications of monosaccharide-azobenzene SAMs were conducted by in situ deprotection of the thioacetyl/acetyl groups of acetylated Gal-Azo-SH/Man-Azo-SH precursors and

subsequent immersion of the Au electrode to the 1 mM ethanolic solution of mixed thiol-freed Gal-Azo-SH/Man-Azo-SH and nhexanethiol (1:10, molar ratio) at room temperature for ∼36 h in dark. The formed monolayer electrode was then rinsed extensively with ethanol and dried in a stream of dry, high-purity nitrogen. To show that the monosaccharide-azobenzene ligands were completely deprotected before grafting on the electrode, time of flight electrospray ionization (TOF-ESI) mass analyses were carried out for the characterization of Gal-Azo-SH/Man-Azo-SH with fully deacetylated hydroxyls and thiols (Figure S2). Photoregulation of the formed Azo-SAMs was carried out by irradiating the electrode immersed in phosphate-buffered saline (PBS, 0.1 M, pH 7.0) with alternate UV (365 nm) and visible (>450 nm) lights. CV, DPV and Faradaic impedance experiments were carried out in a [Fe(CN)6]3−/4− (1.0 mM) + KCl (0.1 M) solution. The CV spectra were scanned from −0.2 to 0.7 V (scan rate: 0.1 V s−1) and the C


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Figure 2. Cyclic voltammetry (CV) of Gal-Azo-SH (A) and Man-Azo-SH (C) modified electrode monolayers (scan rate, 100 mV; 1.0 mM [Fe(CN)6]3−/4− + 0.1 M KCl) after UV/vis irradiation. Differential pulse voltammetry (DPV) of Gal-Azo-SH (B) and Man-Azo-SH (D) modified electrode monolayers (1.0 mM [Fe(CN)6]3−/4− + 0.1 M KCl) after UV/vis irradiation. DPV spectra were taken from 0 to 0.6 V. The Faradaic impedance spectra were taken from 1 to 100 000 Hz. Lectin Interaction. SAM-modified gold electrodes (Azo-SAMs) were first incubated for 20 min in the presence of various concentrations of lectins (from 0.1 to 10 μM) in PBS buffer (0.1 M, pH 7.4) and then rinsed with PBS buffer (0.1 M, pH 7.4) and Milli-Q water. For lectins detachment, the above electrode were immersed in PBS buffer (0.1 M, pH 7.4) and exposed to 365 nm UV light for 15 min and then rinsed with PBS buffer (0.1 M, pH 7.4) and Milli-Q water. Subsequent exposing this electrode to 450−700 nm visible light for 30 min in PBS buffer (0.1 M, pH 7.4) regenerated the trans-AzoSAMs from cis-Azo-SAMs. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and Faradaic impedance experiments were carried out in a [Fe(CN)6]3−/4− (1.0 mM) + KCl (0.1 M) solution. The CV spectra were scanned from −0.2 to 0.7 V (scan rate, 0.1 V s−1), and the DPV spectra were taken from 0 to 0.6 V. The Faradaic impedance spectra were taken from 1 to 100 000 Hz. Cell Culture. Hep-G2 cells were obtained from ATCC (Rockville, MD, U.S.A) and were cultured in a Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, U.S.A.) supplemented with a 10% fetal bovine serum (FBS; Gibco, Gland Island, NY, U.S.A.) at 37 °C in a 5% humidified CO2 air environment. RAW264.7 macrophage cells were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. The cells were grown in 1640-RMPI (Gibco) supplemented with 10% FBS (Gibco), in a humidified atmosphere at 37 °C with 5% CO2. To produce M2 macrophages, the cells were treated with IL-4 (20 ng mL−1) in the growth medium for 24 h.52 Cell Adhesion. SAM-modified gold electrodes (Azo-SAMs) were first incubated for 30 min in the presence of various concentrations of live cells (from 5 000 to 500 000 cells mL−1) in PBS buffer (0.1 M, pH 7.4) and then rinsed with PBS buffer (0.1 M, pH 7.4) and Milli-Q water. For cell detachment, these electrodes were immersed in PBS buffer (0.1 M, pH 7.4) and exposed to 365 nm UV light for 20 min and then rinsed with PBS buffer (0.1 M, pH 7.4) and Milli-Q water.

Subsequently exposing this electrode to 450−700 nm visible light for 30 min in PBS buffer (0.1 M, pH 7.4) regenerated the trans-Azo-SAMs from cis-Azo-SAMs. DPV and Faradaic impedance experiments were carried out in a [Fe(CN)6]3−/4− (1.0 mM) + KCl (0.1 M) solution. The DPV spectra were taken from −0.2 to 0.7 V. The Faradaic impedance spectra were taken from 1 to 100 000 Hz.

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RESULTS AND DISCUSSION The Gal-Azo-SH/Man-Azo-SH self-assembled monolayer (SAM) was fabricated by immersing the electrode in an ethanolic solution of mixed Gal-Azo-SH/Man-Azo-SH ligand and n-hexanethiol (total concentration 1 mM, molar ratio 1:10) at room temperature in the dark for 36 h. The supporting nhexanethiol was added in case the molecular switches assembled on the packed SAMs would lose the free volume for isomerization.53−56 Hence, the supporting n-hexanethiol plays a role as to dilute the grafted Gal-Azo-SH/Man-Azo-SH ligand and make room for efficient isomerization and receptor adhesion. The formation of Gal-Azo/Man-Azo SAM on gold electrodes were first confirmed by TOF-SIMS analysis (Figure 1). Although no obvious molecular peaks of Gal-Azo-SH (572.6570) and Man-Azo-SH (585.6771) were shown on the spectra, probably because of the unstable feature of aliphatic alcohol groups on the saccharides during ion impacts, several characteristic fragment ions were found and assigned, suggesting the monolayer formation. Elemental composition analysis of the SAMs from X-ray photoelectron spectrometry (XPS) further determined the attachment of Gal-Azo-SH and Man-Azo-SH to the gold surfaces (Figure S3). The electrochemistry of the resulting surfaces were then tested via cyclic voltammetry (CV), differential pulse voltammetry (DPV), and D


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Figure 3. DPV response of trans-Gal-Azo SAM (A) and cis-Gal-Azo SAM (B) upon addition of 7 μM PNA (1.0 mM [Fe(CN)6]3−/4− + 0.1 M KCl). DPV response of trans-Man-Azo SAM (C) and cis-Gal-Azo SAM (D) upon addition of 7 μM Con A (1.0 mM [Fe(CN)6]3−/4− + 0.1 M KCl). Percentage (%) of peak current decrease of trans-/cis-Gal-Azo SAM (E) and trans-/cis-Man-Azo SAM (F) upon addition of various selective or unselective proteins (7 μM, 0.1 M PBS buffer, pH 7.4).

Faradaic impedance spectroscopy using [Fe(CN)6]3−/4− as the redox probe (Figures 2 and S16). Compared to the bare electrode, a decreased peak current (E = 0.20 V vs SCE) on CV and DPV spectra and a corresponding increase in the capacitive loop (charge resistibility) were observed for the Gal-Azo/ManAzo SAMs, suggesting an electron hindrance due to the attachment of the ligand to the surface. In view of the excellent trans−cis photoisomerization of these monosaccharide-azobenzene derivatives in solution (Figure S4), photoswitching operations on Gal-Azo/Man-Azo SAMs were conducted under alternate UV/visible light. CV and DPV were used to follow the entire switching process (Figure 2 A−D). Compared to the trans-Azo SAMs (E = 0.20 V vs SCE), the cis-Azo SAMs showed a relatively lower current signal, ∼38.5% of that for the trans-Azo SAMs. The surface-bound cis-Azo structure caused more electron hindrance than the trans-isomer and a less

effective diffusion of [Fe(CN)6]3−/4− to the electrode surface. After the trans−cis photoisomerization, the hydrophobic azobenzene moiety was exposed instead of the originally protruded hydrophilic saccharide (Scheme 1B), thereby partially blocking the diffusion of [Fe(CN)6]3−/4− to the electrode surface. Besides, the significantly increased dipole moment for cis-azobenzene (μ = 4.4 debye for the cis vs 0 debye for trans)18 might be another reason for blocking the efficient diffusion of [Fe(CN)6]3−/4− to the surface. As the varied position of the functional group after trans−cis photoisomerization would affect the hydrophilicity of the modified surface,56,57 to prove our hypothesis, the photoswitchable wettability was further measured (Figure S5). The contact angle of an aqueous buffer droplet placed on the monolayer modified electrode was switched from 53.6 ± 1.1° (trans) to 60.7 ± 1.0° (cis) for the Man-Azo SAM, and from 52.6 ± 1.3° (trans) to E


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Figure 4. DPVs of trans-Gal-Azo SAM (A) and cis-Gal-Azo SAM (B) upon addition of Hep-G2 cells (100 000 cells/mL, 1.0 mM [Fe(CN)6]3−/4− + 0.1 M KCl). DPVs of trans-Man-Azo SAM (C) and cis-Gal-Azo SAM (D) upon addition of M2 macrophage cells (100 000 cells/mL, 1.0 mM [Fe(CN)6]3−/4− + 0.1 M KCl). Percentage (%) of peak current decrease of trans-/cis-Gal-Azo SAM (E) and trans-/cis-Man-Azo SAM (F) upon addition of various selective and unselective cells (100 000 cells/mL, 0.1 M PBS buffer, pH 7.4) (***P < 0.001; **P < 0.05).

61.1 ± 1.1° (cis) for the Gal-Azo SAM. These results suggest that the trans−cis photoisomerization of the Gal-Azo/Man-Azo leads to the hydrophilicity change of the surface, which is consistent with the different saccharide-layer orientations before and after photoswitching. With these photoresponsive SAMs in hand, their “active/ inactive” photoswitching and recognition of selective lectins were carried out. To test our speculation that SAMs with different photoisomeric ligands would possess different activities toward a selective receptor, both monosaccharideazobenzene SAMs were first passivated to the cis-Gal-Azo/ Man-Azo state under UV light (λ = 365 nm) irradiation for 15 min to saturation. The proposed low binding affinities of the dormant cis-Gal-Azo/Man-Azo SAMs were demonstrated through DPV after interacting with several selective (PNA with the Gal-Azo SAM and Con A with the Man-Azo SAM, 7

μM in 0.1 M PBS, pH 7.4) and unselective proteins (lysozyme and bovine serum albumin, 7 μM in 0.1 M PBS, pH 7.4). We observed a weak current change of the cis-Gal-Azo or cis-ManAzo SAMs with the selective lectins and almost no current change of the SAMs with unselective proteins (Figure 3B and D; Figures S7 and S8). This suggests that the cis-Gal-Azo/ Man-Azo SAMs are inert to protein binding. The proposed working mechanism is illustrated in Scheme 1B. The different binding affinities toward proteins may be because of the orientation dependence of the trans-/cis-azobenzene ligands in an interfacial environment.33,56 On one hand, the sugar endgroups switched to a position that is probably less favorable for effective interactions with the specific lectins. On the other hand, the cis-azobenzene structure exposes the nonrecognitive azobenzene moieties toward the added proteins and meanwhile causes the targeting monosaccharide to approach the F


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The photoswitchable recognition of both monosaccharideazobenzene SAMs were also investigated with Faradaic impedance spectrometry. Similarly, we observed that the capacitive loop (charge resistibility) increased significantly in the trans-SAMs with only a selective lectin, whereas no obvious variations were seen for unselective proteins (Figure S17). For the cis-SAMs, the capacitive loop increased much less significantly after interacting with selective lectins, and almost no signal changes were detected for unselective proteins (Figure S18). These results together suggest an “active− inactive” photoconvertible feature of our smart surfaces: that the trans-state of the monosaccharide-azobenzene modified surface has a sufficiently high binding specificity toward selective lectins and could then be “switched off” after converting the trans-state to the cis-state, leading to a weakened affinity toward biomacromolecules. Next, the ability of the photoresponsive smart surfaces functionalized with Gal-Azo-SH/Man-Azo-SH ligands to bind live cancer cells that express sugar receptors were interrogated. Human Hep-G2 cells that overexpress the galactose-selective ASGPr and mouse RAW264.7 (R264.7) cells that can be induced to M2 macrophages with an upregulation in MR expression (a mannose-selective receptor that promotes tumorigenesis and tumor metastasis) were employed. The DPV results shown in Figure 4 suggest an excellent celladhesion ability and selectivity of monosaccharide-transazobenzene associated photoswitchable surfaces. Galactose attached trans-Gal-Azo SAM showed selective interaction with the ASGPr-expressing Hep-G2 cells, with much less interaction with the control HeLa cells without ASGPr expression (Figures 4A and S13).42,46,47 Likewise, the mannose-attached trans-Man-Azo SAM showed a selective interaction with the MR-expressing M2 cells, with low affinity to the control Raw264.7 cells without MR (Figures 4C and S13).46 The percentage of decreased current was subsequently calculated (Figure 4E and F). Compared with the trans-SAMs, a low cell-adhesion ability was observed for both cis-SAMs (Figure 4B and D and Figure S13), suggesting their inactive state for cell adhesion. A concentration-dependent current quenching carried out with the trans-SAMs showed a good linearity over a range from 5 000 to 500 000 cells/mL in 0.1 M PBS solution at pH 7.4 (Figure S14). The LODs of both trans-SAMs for the selective cells were calculated to be 107 cells/mL for trans-Gal-Azo SAM (with Hep-G2 cells) and 230 cells/mL for trans-Man-Azo SAM (with M2 cells). Faradaic impedance spectroscopy was also employed to investigate the cell adhesion on the photoswitchable electrodes. Sharp increments in the capacitive loops were observed when Hep-G2 and M2 cells were incubated with the trans-Gal-Azo and trans-Man-Azo SAMs, respectively (Figure S20). In contrast, no obvious variations were detected for the cis-Gal-Azo/Man-Azo SAMs with the selective cells (Figure S21). These results suggest the selective cell-adhesion ability of the monosaccharide-trans-azobenzene modified surfaces, which could be “photoswitched off” to relatively inactive cis-surfaces on-demand. In addition, the captured cells could be released to the buffer solution after the trans-SAMs were irradiated again with UV light (λ = 365 nm), forming the dormant cis-SAMs. Subsequently, visible light irradiation regenerated the trans-SAMs, enabling a photoinduced capture and release cycle (Figure S15). It should be noted that previous relevant studies on photoswitchable surfaces mainly focused on their ON/OFF

surrounding 1-hexanethiol arrays. The binding affinity with proteins is thus compromised. The smart monosaccharideazobenzene surfaces could then be activated to work via visible light irradiation (λ > 450 nm, 30 min to saturation). The CVs of trans-Gal-Azo/Man-Azo SAMs in the absence or the presence (7 μM in 0.1 M PBS buffer, pH 7.4) of selective lectins were recorded (Figure S6). For Con A with the ManAzo SAM and PNA with the Gal-Azo SAM, the resulting peak current (E = 0.20 V vs SCE) decreased evidently, whereas minor changes were observed when the lectins were added in a reverse fashion. To investigate the selective recognition between our transGal-Azo/Man-Azo SAMs and lectins in more detail, DPVs were recorded. As shown in Figure 3A and C, addition of lectins (PNA to the Gal-Azo SAM; Con A to the Man-Azo SAM; 7 μM in 0.1 M PBS buffer, pH 7.4) led to a much more significant current (E = 0.20 V vs SCE) quenching to ca. 60% of the original state compared to their dormant cis-isomer counterparts (ca. 28%), demonstrating that the trans-surface is more sensitive to selective lectins than the cis-counterpart. The biospecificity of these monosaccharide-Azo SAMs was further tested by adding several other proteins to the electrodes. We observed minor current signal changes of the electrodes after interacting with the unselective proteins (Figures S9 and S10). The percentage of decreased current (Id = (I0 − Is)/I0, where Id is the fractional decrease in current, I0 is the current without lectin, and Is is the current with an added lectin) was subsequently measured for comparison of both the recognition specificity and responsiveness between the active trans-Gal-Azo/Man-Azo SAMs and inactive cis-Gal-Azo/ Man-Azo SAMs with selective and unselective proteins (Figure 3E and F). A >2-fold difference in recognition efficiency was observed between the cis-isomer modified SAMs (cis-SAMs) and the trans-isomer modified SAMs (trans-SAMs) toward selective lectins, illustrating a photoswitchable binding activity of the monosaccharide-azobenzene SAMs. More importantly, it should be noted that the binding affinities toward unselective proteins on the cis-SAMs are further reduced to be almost negligible compared to the working trans-SAMs. This dormant feature toward unselective proteins endows the photoresponsive cis-SAMs with potential anti-fouling properties, which could avoid the unwanted or unnecessary contaminations during transportation and storage. The captured lectins could be released to the buffer solution after the trans-SAMs were irradiated again with UV light (λ = 365 nm), forming the dormant cis-SAMs. Subsequently, visible light irradiation regenerated the trans-SAMs, enabling a photoinduced “capture and release” cycle (Figures S11 and S19). Conventional photoactivate surface materials are mainly based on the photocaged functional groups (e.g., ortho-nitrobenzyl),31,58−61 which are not recyclable once activated by cleaving the caged groups. Surfaces modified with photoswitchable compounds offer great opportunity to fabricate reusable surface/interface materials for biomedical applications, as in this work the activated and deactivated states could be reversibly interconverted under alternate light irradiation. Good linearity for the lectin detection was produced over a range from 0.1 to 10 μM in 0.1 M PBS solution at pH 7.4 (Figure S12). The limits of detection (LODs) of both trans-SAMs were calculated to be 113 nM for trans-Gal-Azo SAM and 103 nM for trans-ManAzo SAM (3σb/k, where σb is the current intensity in the absence of a lectin). G



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adhesion or recognition abilities for targeted molecules or live cells. The working/active ON-state was always highlighted, while the potential application of the OFF-state was less exploited.33−38 Here, we pointed out that the less-active cis-Azo SAMs would play a role as anti-fouling protection prior to functioning on account of their negligible recognition toward unspecific proteins and cells. Although the monosaccharide modified trans-Azo SAM surfaces possess specific recognition and interaction ability with targeted lectins and live cells, certain amounts of unspecific proteins or live cells are inevitably attached to the functional surfaces. Detection errors would thus occur during practical use. The low-adhesive cis-Azo SAM surfaces, on the other hand, further decrease the interaction capabilities with unspecific proteins and liver cells and prevent the functional surfaces from unwanted contamination and consumptions. Moreover, compared with previous related research with monosaccharides as a ligand group,33−35,38 here we extended the scope of photocontrolled biological analyses from bacteria to macrophage and cancer cells. We should also admit that our system is far from perfect. The inherent thermoreversibility of the cis-azobenzene hampers its practical use for long-time storage as the dormant state. Besides, the incomplete trans−cis conversion would be a limitation for accurate detection in more complex or high-accuracydemanding working conditions. We envision that the recently developed novel high thermostable and quantitative isomerizable azobenzene derivatives (half time for years!)62 and highconversion P-type photochromic materials (which are inherently thermo-irreversible)4,63 would be more suitable candidates for carrying out practical tasks in smart functional surfaces.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Junji Zhang: 0000-0003-2823-4637 He Tian: 0000-0003-3547-7485 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by the 973 project (2013CB733700), the National Natural Science Foundation of China (21402050, 21420102004, and 21572058), the Programme of Introducing Talents of Discipline to Universities (B16017), and the Shanghai Rising-Star Program (16QA1401400 to X.-P.H.). Prof. Jia Li and Prof. Yi Zang at Shanghai Institute of Materia Medica are warmly thanked for their help in cellular experiments. Dr. Xin Hua is warmly thanked for her help in TOF-SIMS.

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REFERENCES

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CONCLUSION To summarize, we have developed two photocontrollable monolayer electrode surfaces functionalized with galactoseazobenzene (Gal-Azo-SH) and mannose-azobenzene (ManAzo-SH) ligands. These two smart surfaces showed the ability to take orders from different light signals to operate controllable recognition of selective lectins and cells. Importantly, these smart surfaces could be kept in an inactive state that shows low binding affinities to analytes in their cisisomer structures. This may avoid unwanted contamination prior to use. Further, the trans-SAMs with high binding affinity to analytes could be activated via visible light on demand. Although the thermoreversible property of azobenzene derivatives disfavors the long-time storage of the inactive cisSAMs, this study might provide a new concept for the development of portable and low-cost devices suitable for biomacromolecular and cellular analyses. Our ongoing research focuses on designing thermostable photoswitching smart surfaces with intrinsic electroactive reporters.

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Research Article

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b15599. Additional figures cited in this article, synthesis and characterization of new compounds, and original spectral copies of new compounds (PDF) H


Research Article

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